The objective of this proposal is to elucidate the mechanisms whereby diabetes impairs anestheticinduced preconditioning (ARC) with the goal of reducing perioperative cardiovascular morbidity and mortality in high-risk patients. A growing body of evidence implicates endothelial nitric oxide synthase (eNOS)- derived NO- and downstream effects on mitochondria! function and adenosine triphosphate-regulated potassium (KArp) channels as critical mediators of ARC. The current proposal will test the hypothesis that diabetes and hyperglycemia impair ARC signal transduction mechanisms in the heart through an eNOS-sensitive pathway involving HSP90, tetrahydrobiopterin (BH4), asymmetric dimethylarginine (ADMA), KATp channels, and mitochondria; and that this pathway can be favorably modulated with pharmacological and genetic strategies targeted at NO* signaling and mitochondrial function (mitochondrial genome switch). During Specific Aim 1, we will evaluate the hypotheses that ARC enhances NO signaling by promoting eNOS coupling (production of NO- and not superoxide anion) through mechanisms that involve decreased production of ADMA, increased availability of BH4, and enhanced interactions between HSP90 and eNOS. We propose that ARC improves mitochondrial bioenergetics (ATP production, respiration, mitochondrial membrane potential) after myocardial ischemia and reperfusion, and enhances KATP channel activity in an NO* dependent fashion, and conversely, that these beneficial effects are abolished by diabetes and hyperglycemia. During Specific Aim 2, we will address the hypotheses that targeting specific components of ARC signal transduction pathways by using pharmacological and genetic strategies to improve eNOS function and enhance mitochondrial bioenergetics will restore ARC protection during diabetes. The proposal will exploit a novel model of type 2 diabetes in the rat in which we are able to selectively switch the mitochondrial genome in order to further dissect the role of mitochondria during impaired ARC. Exciting preliminary data indicate that ARC is restored in diabetic rats after mitochondrial genome switch. These experiments will provide novel mechanistic information on the role of eNOS regulation, mitochondrial function, diabetes, and hyperglycemia to modulate anestheticinduced cardioprotection through protein-protein interactions; protein phosphorylation/tyrosine nitration; altered co-factor availability and ROS formation; and through decreased KATP channel activity using novel genetic and pharmalogical approaches in vivo and in engineered heart tissue, isolated hearts, and cells in vitro. The results will also suggest new therapeutic targets for intervention in patients with diabetes. Lay description: Individuals with diabetes are at increased risk for cardiovascular complications following anesthesia and surgery. The proposed research will define the mechanisms whereby diabetes impairs the cardioprotective effects of anesthetics and will identify new potential treatments for diabetes and hyperglycemia.